Studies of the molecular mechanisms underlying neoplastic transformation and progression have resulted in the understanding that cancer is a genetic disease, deriving from the accumulation of a series of acquired genetic lesions. Despite advances in chemotherapy, radiation delivery, and surgical treatment regimens, survival from many advanced cancers remains poor, and it is apparent that alternative treatment approaches are necessary. Gene therapy/virotherapy is a promising strategy for the treatment of cancer. Although attempted in the past and abandoned because of toxicity and inefficacy, the virotherapy approach has reemerged with great promise, in large part due to better understanding of virus biology and the ability to genetically modify viruses. With this knowledge, we can now design viruses to better replicate in and specifically kill tumor cells. Using conditionally replicative adenoviruses (CRAds) represent a method to achieve efficient tumor cell oncolysis and mitigate tumor cell infection limitations. Ideally, cancer-specific replication of CRAds would result in viral-mediated oncolysis of infected tumor tissues and release of the virus progeny, capable of further propagating in surrounding tumor cells but not in those of normal tissues, which would be refractory to CRAd replication. In our Phase I study, we exploited a novel cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of a CRAd virotherapy agent. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. The technical and scientific merit and feasibility of this approach that combines both transcriptional and translational regulation strategies for the key goal of CRAd replicative specificity has now been established. Our Phase I in vitro and in vivo studies demonstrated strong proof-of-principle that this CRAd agent, utilizing a heterologous mRNA translational control element, retains anti-tumor potency. The objective of this Phase II application is to continue the R&D efforts initiated in Phase I by optimizing the CRAd virotherapy agent with enhanced tumor infectivity. Relative resistance of tumor tissues to adenovirus serotype 5 (Ad5) infections has been noted in a number of in vivo gene therapy trials. Deficiency of the primary Ad receptor CAR, is understood to be the biologic basis of this phenomenon. The inclusion of a high affinity Arg-Gly-Asp (RGD) motif into the HI loop of the Ad5 fiber knob has been shown to overcome low CAR expression on tumor cells, thereby enhancing transduction. In Specific Aim 1, cancer cell transduction of the CRAd virotherapy agent will be enhanced by inserting an RGD motif. In Specific Aim 2, in vivo experiments will be conducted to evaluate the biological efficacy and establish specific treatment protocols in animal models of loco-regional and disseminated disease. Finally, in Specific Aim 3, biodistribution and toxicology studies required for IND submission to the FDA will be performed. Successful completion of this Phase II application will lead directly to clinical investigation of the modified CRAd agent (CXCR4-5'-UTR-RGD CRAd) in patients with disseminated disease. PUBLIC HEALTH RELEVANCE: Despite many advances in standard treatment regimens, survival from many advanced cancers remains poor, and it is apparent that new alternative treatment approaches are necessary. Using viruses designed to divide in and specifically kill tumor cells has become a promising approach to better understanding of virus biology and the ability to genetically modify viruses. The objective of this application is to further develop a conditionally replicative adenovirus (CRAd) that has been designed to infect, divide and lyse efficiently in tumor cells but not in normal cells;successful completion.